Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization

A new method for the analysis of ubiquinones in various samples was developed using an HPLC system with postcolumn derivatization. Craven's reaction, a specific color reaction for the analysis of ubiquinones, was used in the system. Because the reaction progressed in organic solvents that contained ubiquinones and ethylcyanoacetate under an alkaline condition, the selectivity for ubiquinone detection was higher than that for ubiquinone detection using the nonderivatized ultraviolet detection system at 275 nm, a system widely used for the analysis of ubiquinones. The new detection system can avoid the adverse effects of impurities. Furthermore, it can confirm specificity by stopping the color reaction under a neutral condition. The detection limit for ubiquinone-10 was 1 ng (1.2 pmol). A good linearity for the calibration curve was observed in the range of 11.7 pmol to 11.7 nmol. To investigate the possible application of this method, various samples, such as soybean capsules used as a dietary supplement and biological materials (rice as well as bovine plasma and liver samples), were applied to the system and their ubiquinone contents were quantified. This method is thought to be widely and conveniently applicable for determining the level of ubiquinones because of its high selectivity for ubiquinone detection.     

High-performance liquid chromatography of fatty acid isopropylidene hydrazides and its application in lipid analysis

Fatty acid isopropylidene hydrazides, prepared by stepwise treatment of acyl lipids with hydrazine and acetone, were analyzed by high-performance liquid chromatography on a reversed-phase column. These derivatives could be easily eluted with 15% water in methanol and monitored by measuring absorbance at 229 nm with a uv detector. Their elution behavior, in general, was similar to that of methyl esters and some commonly used ultraviolet-absorbing derivatives of fatty acids. The new method has been used for fatty acid analysis of some oils.     

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

The purpose of the study reported here was to develop a method for the determination of lipid classes in intestinal fluids, including bile acids (BAs). A solid-phase extraction (SPE) method using C18 and silica columns for the separation of BAs, phospholipids (PLs), and neutral lipids (NLs), including free fatty acids, has been developed and validated. Fed-state small intestinal fluid collected from humans was treated with orlistat to inhibit lipolysis and mixed with acetic acid and methanol before SPE to maximize lipid recoveries. BAs, PLs, and NLs were isolated using lipophilic and polar solvents to promote elution from the SPE columns. The different lipid classes were subsequently analyzed using three separately optimized HPLC methods with evaporative light-scattering detectors. High recoveries (>90%) of all lipids evaluated were observed, with low coefficients of variation (<5%). The HPLC methods developed were highly reproducible and allowed baseline separation of nearly all lipid classes investigated. In conclusion, these methods provide a means of lipid class analysis of NLs, PLs, and BAs in human fed-state small intestinal fluid, with potential use in other fluids from the intestinal tract and animals.     

Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50-100 microl) and minimal sample pretreatment. A Hypersil BDS RP-C(18) column (250 x 4.6 mm, 5 microm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5-500 microM. Limits of detection and quantification were in the range of 0.07-0.60 microM and 0.20-1.80 microM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation

A commonly used method for quantification of lipid peroxidation depends upon measurement of a malonaldehyde-thiobarbituric acid derivative with absorbance at 532 nm. Investigation of this assay demonstrated that erythrocyte peroxidation produces compounds that react with thiobarbituric acid to interfere with the malonaldehyde assay. Interference results from carryover absorbance at 532 nm, equivalent to 20% of the intensity of the maximum absorption peak at 453 nm. These compounds are not products of lipid peroxidation but are derived from erythrocyte hemolysate and reduced glutathione. A specific HPLC assay for malonaldehyde corroborated the improved accuracy of measuring absorbance at 453 nm and correcting for the absorbance of the interfering compounds at 532 nm when assaying erythrocyte malonaldehyde production.     

Fluorometric determination of guanidino compounds by new postcolumn derivatization system using reversed-phase ion-pair high-performance liquid chromatography

A new postcolumn derivatization system using 1,2-naphthoquinone-4-sulfonate as fluorogenic reagent for the fluorometric determination of guanidino compounds is described. The guanidino compounds were separated by reversed-phase ion-pair high-performance liquid chromatography with an isocratic mobile phase containing the fluorogenic reagent and octane-sulfonate as the counterion. Fluorophors were derived from a condensation of guanidino compounds with the fluorogenic reagent in an alkaline solution. The chromatographic system using the mobile phase containing the fluorogenic reagent was simplified because only two pumps were required to deliver the mobile phase and the alkaline solution. Separation of guanidino compounds was completed within 25 min using a Nucleosil C8 column (5 microns, 15 cm X 4.6 mm i.d.). This method was applied to serum obtained from patients on hemodialysis therapy.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids

The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.     

HPLC测定灵芝子实体水提物及相关产品中三萜的含量

灵芝药品大多以灵芝子实体水提物为原料,为快速准确测定灵芝子实体水提物及相关产品中三萜的含量,建立了具有较好分离效果的HPLC分析测定方法。通过优化色谱柱和洗脱条件,优选出Agilent Zorbax SB-Aq C18色谱柱(250 mm×4.6 mm,5μm),以乙腈-乙酸水溶液(0.01%)为流动相梯度洗脱,流速为1.0 mL/min,检测波长252 nm,柱温30℃,该条件下灵芝酸A、灵芝酸F等10种灵芝酸得到较好的分离。方法学考察显示该分析方法精密度、重复性、稳定性和加样回收率的RSD值均小于5%,可以用于灵芝酸C2、灵芝酸G、灵芝烯酸B、灵芝酸B等10种灵芝酸的定量检测。通过对灵芝子实体原料、水提物和市售灵芝产品中10种三萜类成分分析发现,灵芝子实体水提物中均含有这10种三萜,含量为2.52%–6.83%,较子实体原料大幅提高,市售的灵芝产品中的三萜含量为0.27%–0.84%。该方法的建立为灵芝水提物及其产品质量标准的建立奠定基础。     

A specific,accurate, and sensitive measure of total plasma malondialdehyde by HPLC

Malondialdehyde (MDA) is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid (TBA) with MDA to yield a pink chromogen attributable to an MDA-TBA₂ adduct is a common assay approach with products being quantified by ultraviolet-Vis assay as nonspecific TBA-reactive substances (TBARS) or chromatographically as MDA. The specificity of the TBARS assay was compared with both chromatographic assays for total plasma MDA. The levels of total plasma MDA were significantly lower than the plasma TBARS in each of the samples examined, and interestingly, the interindividual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the four online chromatographic detectors yielded a precise, sensitive, and accurate determination of total plasma MDA, and selected-ion monitoring was the most-accurate assay (101.3%, n = 4). The online diode array detectors provided good assay specificity (peak purity index of 999), sensitivity, precision, and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays, which claim to quantify MDA, and it is proposed that the TBARS approach may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.     

Automated analysis of uronic acids by high-performance liquid chromatography with photometric and fluorimetric postcolumn labelling using 2-cyanoacetamide

A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

A comparison of anion-exchange and steric-exclusion HPLC assays of mouse plasma lipoproteins

We compared two HPLC methods (anion exchange [AE] and steric exclusion [SE]) for analysis of mouse lipoprotein profiles by determining coefficients of variability (CVs) under varying conditions. CVs for AE and SE were comparable on fresh samples. There was an inverse relationship between subfraction curve area and CV [r = -0.65 (AE) and -0.50 (SE)], consistent with the interpretation that as curve area decreased, error variance increased and signal-to-noise ratio decreased. Sample storage did not affect SE. In contrast, with AE, alterations in measured lipoproteins were apparent after storage, including a decrease in the HDL subfraction [66.8% (baseline) vs. 15.9% (1 week); P < 0.01] and an increase in areas under LDL and VLDL peaks. Concomitant with decreasing HDL area, reproducibility deteriorated with the duration of storage. Analysis of the effects of decreasing sample injectate volume to <25 microl on SE lipoprotein subfractions revealed that areas under LDL and VLDL peaks decreased and persisted as volume was decreased further. Areas under all lipoprotein subfractions measured with either AE or SE were linearly correlated with the amount of cholesterol [r = 0.69 (AE) and 0.87 (SE)]. Both AE and SE yield reproducible, accurate, and rapid measurements of lipoproteins from small amounts of serum. AE yields more sensitive, high-amplitude, well-defined peaks that can be easily distinguished and necessitates the use of smaller sample volumes compared with SE, but sample storage causes alterations in the chromatogram. SE appears better suited to serial analyses involving stored samples.     

HPLC法测定不同产地和品种柠檬果皮中柠檬苦素和诺米林含量

该研究建立了同时测定檬苦素和诺米林含量的高效液相色谱法,并采用该方法对8种不同柠檬样品中柠檬苦素和诺米林含量进行了测定。色谱条件如下:安捷伦Eclipse XDB-C18(250 mm×4.6 mm,5μm)色谱柱,流动相为0.1%磷酸水溶液(A)-乙腈(B),梯度洗脱,流速为1.0 mL·min^-1,柱温为30℃,波长为210 nm。结果表明:3个产地尤力克果皮中柠檬苦素含量依次为云南瑞丽(EUR_D)、四川安岳(EUR_C)和广东河源(EUR_Y),诺米林含量依次为EUR_D、EUR_C和EUR_Y;不同品种柠檬果皮中柠檬苦素含量依次为北京柠檬(M_NM)、粗柠檬(R_NM)、无核柠檬(S_NM)、热那亚(RNY)、里斯本(LSB),诺米林含量依次为S_NM、R_NM、LSB、RNY和M_NM。综上结果表明,不同产地和品种柠...     

High-performance liquid chromatography (HPLC) determination of inosine, a potential biomarker for initial cardiac ischaemia, using isolated mouse hearts

Each year in the USA approximately 7-8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2-5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs-Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C₁₈ column at a flow rate of 0.6 ml min^-1 with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml^-1 using a 100-µl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.     

High-performance liquid chromatography (HPLC) determination of inosine,a potential biomarker for initial cardiac ischaemia,using isolated mouse hearts

Abstract

Each year in the USA approximately 7–8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2–5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs–Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C₁₈ column at a flow rate of 0.6 ml min^?1 with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml^?1 using a 100-µl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.